Re: [ccp4bb] Strange diffraction image
Could be an organic crystal - what's the resoution of the lowest order reflections? Jan D. On Fri, Oct 12, 2012 at 8:11 AM, Chang Qing robie0...@gmail.com wrote: Hi, everyone: I just got some strange diffraction images from crystals with triangular pyramid shape. I think this should not be protein diffraction. I never saw so strange images like this. Does anyone know what it is? Is it a kind of salt diffraction? Thank you very much Chang -- Jan Dohnalek, Ph.D Institute of Macromolecular Chemistry Academy of Sciences of the Czech Republic Heyrovskeho nam. 2 16206 Praha 6 Czech Republic Tel: +420 296 809 340 Fax: +420 296 809 410
Re: [ccp4bb] On maps and doubts
Have you got two very similar datasets (isomorphous) inbetween which the mentioned difference should be pronounced? You could try a cross-crystal difference map as well. Jan On Fri, Oct 5, 2012 at 8:40 AM, herman.schreu...@sanofi.com wrote: ** Dear Israel, I wonder why you do not see anything in your Fo-Fc (mFo-DFc?) maps, since the limit for a (n+1) * mFo - n * DFc map, when n approaches inifinity, is the mFo-DFc difference map. There is nothing wrong in going further like with 4mFo-3DFc maps, but you should bear in mind that they are becoming more and more difference map-like and less and less regular maps. What I think is happening is that since you added your ribosomal factor to preformed crystals, you have incomplete occupancy, say the factor bound only to 50% or less of the ribosomes. This means that you have to go down in contour level from the 3 sigma which is the default for difference maps to maybe 1.5 or even 1 sigma. If the factor is bound indeed at lower level you will see relatively nice density, if nothing is there, you will see only noise. To prove a conformational change at a single residue, I would just leave it out and refine. Since the refinement program has no reason to bias either conformation, the result should be unbiased. However, in the case of partial occupancy, the resulting map will be a superposition of both conformations. Also to reduce model bias, you might want to try the Buster program. Best regards, Herman -- *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *Israel Sanchez *Sent:* Thursday, October 04, 2012 9:17 PM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] On maps and doubts Hello everyone, I would like to share my experience with one dataset and request some advice on which is the best way to prove a conformational change seen in a density map. The first issue arose when we were looking for an extra ribosomal factor added to a crystalized ribosome. After careful data collection and refinement (I/sigma last shell 1.2, 3.1A and CC1/2 around 22%) the sigma-A-weighted maps 2mFo-DFc and Fo-Fc does not show any clear difference density that we could interpret as the expected factor. Interestingly, a computed map with coefficients 3mFo-2DFc started to show some features that clearly could be explained as a fragment of the factor. The density improved even more with a B-sharpened map. We have seen this behavior before and I was wondering if someone else is using this kind of maps and may could explain the reason behind this density improvement. Is it a crazy idea to go even higher like 4mFo-3DFc? The second query has to do with which is the best way to prove that a conformational change is present in an specific residue (in this case and RNA base) in your structure. To my knowledge, a classic omit map with simulated annealing would do the job regarding removing the model bias. Actually, I found an interesting alternative in PHENIX called a Kick map, were a series of maps computed from a ramdoinised set of models yields a averaged map ideally free from model bias. Does anyone has a preference for any of those schemes? Are there more alternative to prove a conformational change in a model phased with a molecular replacement solution? Thank you very much in advance. -- Israel Sanchez Fernandez PhD Ramakrishnan-lab MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH, UK -- Jan Dohnalek, Ph.D Institute of Macromolecular Chemistry Academy of Sciences of the Czech Republic Heyrovskeho nam. 2 16206 Praha 6 Czech Republic Tel: +420 296 809 340 Fax: +420 296 809 410
Re: [ccp4bb] RE : [ccp4bb] Strange diffraction image
Hi, Thanks for answering my question. I think I'd better provide more informations. These four images were taken from one crystals. The distance of image1-2 is 250mm, and image3-4 is 100mm. I checked more than 10 crystals and results were similar. The spots look very large. The lowest resolution is about 6A. As my protein can hydrolyze ATP, so protein buffer with 5mM of ATP. There is 0.2M of MgCl2 in precipitant buffer with PH7.0. 0.06M of CsCl can improve quality of crystals. I also setup control, in which target protein was not added, and could get nothing in it. I don't think it is a protein crystal. But salt spot should not be so large. And the rings are not just ice-ring. As I got crystal first in hampton crystal screen kit with MgCl2, TrisHCl and PEG4,000, there are only rings in images from 5-6A to about 3A. 2012/10/12 THOMPSON Andrew andrew.thomp...@synchrotron-soleil.fr: Hi Chang No mention of the resolution limit / oscillation range (I think I can see an ice ring, so I would guess 2.5 A?), but it looks like salt to me, with some weaker satellite peaks that may be something weird like an incommensurate phase. Did you try to index? Cheers Andy De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Chang Qing [robie0...@gmail.com] Date d'envoi : vendredi 12 octobre 2012 08:11 À : CCP4BB@JISCMAIL.AC.UK Objet : [ccp4bb] Strange diffraction image Hi, everyone: I just got some strange diffraction images from crystals with triangular pyramid shape. I think this should not be protein diffraction. I never saw so strange images like this. Does anyone know what it is? Is it a kind of salt diffraction? Thank you very much Chang
[ccp4bb] Vitamin B12 (cobalamin) geometry issues
If you are working on a protein that binds vitamin B12 (cobalamin) then you may be interested that there appears to be an issue with geometry of the B12 dictionary currently distributed by ccp4. The problem is that atom C19 in the corrin ring is defined as being SP2, planar with no hydrogen atom attached. Small molecule structures of B12 clearly show this atom is tetrahedral (as do high resolution protein complexes). A survey of 53 PDB structures containing B12 reveals that 19 have C19 atoms that are not sufficiently chiral. All are recent - structures prior to 2008 are all OK. The problem also effects the PDB chemical components definition of B12. Please see https://www.globalphasing.com/buster/wiki/index.cgi?B12Dictionary This page provides a dictionary for B12 with the problem fixed. Hope this proves useful. Regards, Oliver | Dr Oliver Smart | | Global Phasing Ltd., Cambridge UK | | http://www.globalphasing.com/people/osmart/ |
Re: [ccp4bb] RE : [ccp4bb] Strange diffraction image
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Chang, What makes you think spots from salt crystals should not be as large? You have got an ordinary small molecule crystal (or probably a cluster of them) in the beam, with a unit cell somewhat bigger than that of ice judging by the extra real ice rings you've got. Hydrolysis of ATP and Mg2+ present - it's probably (Mg)3(PO4)2- it is very unsoluble http://en.wikipedia.org/wiki/Solubility_table. Best, Tim On 10/12/2012 09:29 AM, Chang Qing wrote: Hi, Thanks for answering my question. I think I'd better provide more informations. These four images were taken from one crystals. The distance of image1-2 is 250mm, and image3-4 is 100mm. I checked more than 10 crystals and results were similar. The spots look very large. The lowest resolution is about 6A. As my protein can hydrolyze ATP, so protein buffer with 5mM of ATP. There is 0.2M of MgCl2 in precipitant buffer with PH7.0. 0.06M of CsCl can improve quality of crystals. I also setup control, in which target protein was not added, and could get nothing in it. I don't think it is a protein crystal. But salt spot should not be so large. And the rings are not just ice-ring. As I got crystal first in hampton crystal screen kit with MgCl2, TrisHCl and PEG4,000, there are only rings in images from 5-6A to about 3A. 2012/10/12 THOMPSON Andrew andrew.thomp...@synchrotron-soleil.fr: Hi Chang No mention of the resolution limit / oscillation range (I think I can see an ice ring, so I would guess 2.5 A?), but it looks like salt to me, with some weaker satellite peaks that may be something weird like an incommensurate phase. Did you try to index? Cheers Andy De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Chang Qing [robie0...@gmail.com] Date d'envoi : vendredi 12 octobre 2012 08:11 À : CCP4BB@JISCMAIL.AC.UK Objet : [ccp4bb] Strange diffraction image Hi, everyone: I just got some strange diffraction images from crystals with triangular pyramid shape. I think this should not be protein diffraction. I never saw so strange images like this. Does anyone know what it is? Is it a kind of salt diffraction? Thank you very much Chang - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFQeAheUxlJ7aRr7hoRAibDAJ9yAfiSwNmh8R4tGwUIwFEZno2qWACfStCM y+xKb+FGGglmv8lTL9Ej8ZQ= =ub/P -END PGP SIGNATURE-
[ccp4bb] EMBO Practical Course in “High throughput Protein Production and Crystallization
Dear All, We are happy to announce an EMBO Practical Course in “High throughput Protein Production and Crystallization” The overall aim of the course will be to review the state-of-the-art in HTP structural biology with an emphasis on methods to study complex targets, including membrane proteins. The course is aimed at early career post-doctoral scientists and PhD students in their second or third year of study. Practical training will be provided in: Structural bioinformatics High throughput cloning expression in E. coli and mammalian cells Protein purification and biophysical characterisation High throughput protein crystallization High throughput data collection at synchrotrons Speakers: Lawrence Kelley - Imperial College London David Drew - Imperial College London Imre Berger - EMBL Grenoble Chris Tate - University of Cambridge Naomi Chayen Imperial College London Opher Gileadi - University of Oxford Renaud Vincentelli - AFMB, Marseille Rob Meijers - EMBL, Hamburg Robert Gilbert - University of Oxford Scott Lesley - Genomics Institute of the Novartis Research Foundation- USA Joanne Nettleship - University of Oxford Louise Bird - University of Oxford Jon Diprose - University of Oxford Martin Walsh - Diamond Light Source Isabel De Moraes - Imperial College, London Venue: Research Complex at Harwell - UK Registration deadline: JANUARY 31ST, 2013 Total Number of Participants limited to: 20 participants For information: http://events.embo.org/13-htp-protein/index.html For Registration: http://events.embo.org/13-htp-protein/application.html http://events.embo.org/13-htp-protein/application.htmlThe organizers Ray Owens (OPPF-UK) Isabel Moraes (MPL) Martin Walsh (Diamond) -- This e-mail and any attachments may contain confidential, copyright and or privileged material, and are for the use of the intended addressee only. If you are not the intended addressee or an authorised recipient of the addressee please notify us of receipt by returning the e-mail and do not use, copy, retain, distribute or disclose the information in or attached to the e-mail. Any opinions expressed within this e-mail are those of the individual and not necessarily of Diamond Light Source Ltd. Diamond Light Source Ltd. cannot guarantee that this e-mail or any attachments are free from viruses and we cannot accept liability for any damage which you may sustain as a result of software viruses which may be transmitted in or with the message. Diamond Light Source Limited (company no. 4375679). Registered in England and Wales with its registered office at Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom
[ccp4bb] NCS mask
Dear CCP4 community, I have a MR solution with 2 mol/asu. The solution looks very good in terms of packing (with reasonable starting R and Rfree) and there are no clashes with the symmetry-mates as well. But when I try to generate a solvent mask I get a truncated mask which covers only 1 and 1/2 molecules. This is irrespective of the program (ncsmask, MAMA and cns) I use to generate the same. NCSMASK gives the following warning. Warning: The mask box covers more than one unit cell Is there anything wrong with the solution or the way in which I am generating mask ? Thanking you in advance for your help. Regards, -Kiran
Re: [ccp4bb] Fwd: [ccp4bb] RE : [ccp4bb] Strange diffraction image
Dear Chang, i have seen ATP diffraction, it's not very different of your image. Maybe you have only ATP in your crystals? Best regard Nicolas Le 12/10/12 14:56, Chang Qing a écrit : Dear Tim I think your explanation is logical. But I tried ADP as ligand first and got crystals and diffraction. ATP in additive kit was found to improve the quality of crystals from cluster to single crystal. CsCl can go on improving the quality and finally I got crystals like this. Is it possible that I get some strange crystals such as CsMgCl3 or something else? Best regard Chang -- Forwarded message -- From: Tim Gruene t...@shelx.uni-ac.gwdg.de Date: 2012/10/12 Subject: Re: [ccp4bb] RE : [ccp4bb] Strange diffraction image To: Chang Qing robie0...@gmail.com 抄送： CCP4BB@jiscmail.ac.uk -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Chang, What makes you think spots from salt crystals should not be as large? You have got an ordinary small molecule crystal (or probably a cluster of them) in the beam, with a unit cell somewhat bigger than that of ice judging by the extra real ice rings you've got. Hydrolysis of ATP and Mg2+ present - it's probably (Mg)3(PO4)2- it is very unsoluble http://en.wikipedia.org/wiki/Solubility_table. Best, Tim On 10/12/2012 09:29 AM, Chang Qing wrote: Hi, Thanks for answering my question. I think I'd better provide more informations. These four images were taken from one crystals. The distance of image1-2 is 250mm, and image3-4 is 100mm. I checked more than 10 crystals and results were similar. The spots look very large. The lowest resolution is about 6A. As my protein can hydrolyze ATP, so protein buffer with 5mM of ATP. There is 0.2M of MgCl2 in precipitant buffer with PH7.0. 0.06M of CsCl can improve quality of crystals. I also setup control, in which target protein was not added, and could get nothing in it. I don't think it is a protein crystal. But salt spot should not be so large. And the rings are not just ice-ring. As I got crystal first in hampton crystal screen kit with MgCl2, TrisHCl and PEG4,000, there are only rings in images from 5-6A to about 3A. 2012/10/12 THOMPSON Andrew andrew.thomp...@synchrotron-soleil.fr: Hi Chang No mention of the resolution limit / oscillation range (I think I can see an ice ring, so I would guess 2.5 A?), but it looks like salt to me, with some weaker satellite peaks that may be something weird like an incommensurate phase. Did you try to index? Cheers Andy De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Chang Qing [robie0...@gmail.com] Date d'envoi : vendredi 12 octobre 2012 08:11 À : CCP4BB@JISCMAIL.AC.UK Objet : [ccp4bb] Strange diffraction image Hi, everyone: I just got some strange diffraction images from crystals with triangular pyramid shape. I think this should not be protein diffraction. I never saw so strange images like this. Does anyone know what it is? Is it a kind of salt diffraction? Thank you very much Chang - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFQeAheUxlJ7aRr7hoRAibDAJ9yAfiSwNmh8R4tGwUIwFEZno2qWACfStCM y+xKb+FGGglmv8lTL9Ej8ZQ= =ub/P -END PGP SIGNATURE-
Re: [ccp4bb] Fwd: [ccp4bb] RE : [ccp4bb] Strange diffraction image
Dear Nicolas ATP crystals is a reasonable answer. Thank you very much. Best regard Chang 2012/10/12 Nicolas Foos nicolas.f...@afmb.univ-mrs.fr: Dear Chang, i have seen ATP diffraction, it's not very different of your image. Maybe you have only ATP in your crystals? Best regard Nicolas Le 12/10/12 14:56, Chang Qing a écrit : Dear Tim I think your explanation is logical. But I tried ADP as ligand first and got crystals and diffraction. ATP in additive kit was found to improve the quality of crystals from cluster to single crystal. CsCl can go on improving the quality and finally I got crystals like this. Is it possible that I get some strange crystals such as CsMgCl3 or something else? Best regard Chang -- Forwarded message -- From: Tim Gruene t...@shelx.uni-ac.gwdg.de Date: 2012/10/12 Subject: Re: [ccp4bb] RE : [ccp4bb] Strange diffraction image To: Chang Qing robie0...@gmail.com 抄送： CCP4BB@jiscmail.ac.uk -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Chang, What makes you think spots from salt crystals should not be as large? You have got an ordinary small molecule crystal (or probably a cluster of them) in the beam, with a unit cell somewhat bigger than that of ice judging by the extra real ice rings you've got. Hydrolysis of ATP and Mg2+ present - it's probably (Mg)3(PO4)2- it is very unsoluble http://en.wikipedia.org/wiki/Solubility_table. Best, Tim On 10/12/2012 09:29 AM, Chang Qing wrote: Hi, Thanks for answering my question. I think I'd better provide more informations. These four images were taken from one crystals. The distance of image1-2 is 250mm, and image3-4 is 100mm. I checked more than 10 crystals and results were similar. The spots look very large. The lowest resolution is about 6A. As my protein can hydrolyze ATP, so protein buffer with 5mM of ATP. There is 0.2M of MgCl2 in precipitant buffer with PH7.0. 0.06M of CsCl can improve quality of crystals. I also setup control, in which target protein was not added, and could get nothing in it. I don't think it is a protein crystal. But salt spot should not be so large. And the rings are not just ice-ring. As I got crystal first in hampton crystal screen kit with MgCl2, TrisHCl and PEG4,000, there are only rings in images from 5-6A to about 3A. 2012/10/12 THOMPSON Andrew andrew.thomp...@synchrotron-soleil.fr: Hi Chang No mention of the resolution limit / oscillation range (I think I can see an ice ring, so I would guess 2.5 A?), but it looks like salt to me, with some weaker satellite peaks that may be something weird like an incommensurate phase. Did you try to index? Cheers Andy De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Chang Qing [robie0...@gmail.com] Date d'envoi : vendredi 12 octobre 2012 08:11 À : CCP4BB@JISCMAIL.AC.UK Objet : [ccp4bb] Strange diffraction image Hi, everyone: I just got some strange diffraction images from crystals with triangular pyramid shape. I think this should not be protein diffraction. I never saw so strange images like this. Does anyone know what it is? Is it a kind of salt diffraction? Thank you very much Chang - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFQeAheUxlJ7aRr7hoRAibDAJ9yAfiSwNmh8R4tGwUIwFEZno2qWACfStCM y+xKb+FGGglmv8lTL9Ej8ZQ= =ub/P -END PGP SIGNATURE-
[ccp4bb] off-topic: detergents for the stabilisation of water-soluble proteins
Hi, Sorry for off-topic question. Does anyone have experience of the stabilisation of water-soluble proteins by detergents? Protein I'm working with is definitely water-soluble and has high yield, but, unfortunately, not very stable. Especially during concentration. So, we thought that adding some detergents may one of the ways to stabilise protein. So, did anyone do it before or may be know published examples? Any suggestions on the detergent type/concentration would be welcome. Thanks, Vitali
[ccp4bb] solubility estimates for domains/structures?
Hi all, Does anyone a program/paper that would give some quantitative estimate for protein solubility based on surface property analysis? (excluding obvious things such as integral membrane / TM regions) Best Tommi
Re: [ccp4bb] off-topic: detergents for the stabilisation of water-soluble proteins
Hi, Yes, it is worth trying. Nonionic detergents can be good for this. One example I know of and readily comes to mind is the use of 0.1% NP40 (Noniondet P40) in the stabilization of murine reverse transcriptase during purification (also helps toprevent precipitation), first described in: Purification and characterization of murine retroviral reverse transcriptase expressed in Escherichia coli., Roth MJ, Tanese N, Goff SP., J Biol Chem. 1985 Aug 5;260(16):9326-35. http://www.ncbi.nlm.nih.gov/pubmed/2410413 DDM may also be good, use at concentration much below CMC. Might be best to check out and screen several detergents, either by mini column purification or by DLS. Thanks, Debanu. -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Vitali Stanevich Sent: Friday, October 12, 2012 9:42 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] off-topic: detergents for the stabilisation of water-soluble proteins Hi, Sorry for off-topic question. Does anyone have experience of the stabilisation of water-soluble proteins by detergents? Protein I'm working with is definitely water-soluble and has high yield, but, unfortunately, not very stable. Especially during concentration. So, we thought that adding some detergents may one of the ways to stabilise protein. So, did anyone do it before or may be know published examples? Any suggestions on the detergent type/concentration would be welcome. Thanks, Vitali
Re: [ccp4bb] solubility estimates for domains/structures?
Hi, Using a surface property analysis (3D structure is available), I think you can get a quantitative estimate by using PISA to find the solvent-accessible area and salvation energy and then comparing that to corresponding values obtained by using in your lab another protein for calibration (maybe ones that you label as highly, medium or minimal soluble with known mg/ml). If you just want an estimate from sequence analysis for solubility on heterologous overexpression, you can try: SOLpro in http://scratch.proteomics.ics.uci.edu/ Or http://mips.helmholtz-muenchen.de/proso/proso.seam http://www.biotech.ou.edu/ Thanks, Debanu -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Tommi Kajander Sent: Friday, October 12, 2012 10:23 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] solubility estimates for domains/structures? Hi all, Does anyone a program/paper that would give some quantitative estimate for protein solubility based on surface property analysis? (excluding obvious things such as integral membrane / TM regions) Best Tommi
Re: [ccp4bb] off-topic: detergents for the stabilisation of water-soluble proteins
The following paper (which can be found at www.wolfson.huji.ac.il/purification/PDF/Literature/Bondos2003.pdf Detection and prevention of protein aggregation before, during, and after purification. Sarah E. Bondos and Alicia Bicknell (2003) Analytical Biochemistry, 316, 223-231 contains a table of agents that may promote protein solubility on the 2nd page. Most are non-detergents which may be worth following up as well as Tween 80, Tween 20 and Nonidet P-40 at the recommended concentrations and their references. Dan
Re: [ccp4bb] solubility estimates for domains/structures?
Hi, Thanks, i was thinking of a situation where one can model the sequence to a structure based on homologous structures, ie assuming i know the fold - i guess PISA / ASA based estimates are the first thing. (mainly indeed the likelyhood to aggregate ie the surface property estimates are what i am looking for.) Tommi On Oct 12, 2012, at 8:38 PM, Das, Debanu wrote: Hi, Using a surface property analysis (3D structure is available), I think you can get a quantitative estimate by using PISA to find the solvent-accessible area and salvation energy and then comparing that to corresponding values obtained by using in your lab another protein for calibration (maybe ones that you label as highly, medium or minimal soluble with known mg/ml). If you just want an estimate from sequence analysis for solubility on heterologous overexpression, you can try: SOLpro in http://scratch.proteomics.ics.uci.edu/ Or http://mips.helmholtz-muenchen.de/proso/proso.seam http://www.biotech.ou.edu/ Thanks, Debanu -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Tommi Kajander Sent: Friday, October 12, 2012 10:23 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] solubility estimates for domains/structures? Hi all, Does anyone a program/paper that would give some quantitative estimate for protein solubility based on surface property analysis? (excluding obvious things such as integral membrane / TM regions) Best Tommi Tommi Kajander, Ph.D., Docent Structural Biology and Biophysics Institute of Biotechnology University of Helsinki Viikinkaari 1 (P.O. Box 65) 00014 Helsinki Finland p. +358-9-19158903 tommi.kajan...@helsinki.fi http://www.biocenter.helsinki.fi/bi/kajander/Kajander_Lab/Home.html
Re: [ccp4bb] off-topic: detergents for the stabilisation of water-soluble proteins
Vitali, Echoing what Dan said, I am not sure why you have chosen detergents first, as there are many other agents which stabilize proteins. Is the instability due to hydrophobic surfaces (e.g., made worse at higher salt) or not. Some non-detergent suggestions are: 1) diols like MPD (see work from Gil Prive's group) 2) non-detergent sulfobetaines (NDSBs), which is the head group in the zwittergent class of deterents. 3) Trimethylamine oxide, which is the head group in the amine oxide class of deterents (LDAO). 4) 200-500 mM L-Arginine 5) Also try 200-500 mM LiCl. The recommendations you received for detergents are very good ones, but remember that many of these detergents are quite dirty, as well as being chemically heterogeneous. Tween 80, Tween 20 and Nonidet P-40 are generally sold at industrial purity (i.e., they are delivered in tanker cars), so either clean them up yourself or buy purified samples (like the Pierce Surfact-Amps brand). Also, be aware that they may be hard to get rid of when you come to setting up crystals. Also look up Sam Gellman's work on detergent-assisted refolding for other stabilizing detergents. Good luck, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334Email: rmgarav...@gmail.com On Oct 12, 2012, at 12:42 PM, Vitali Stanevich wrote: Hi, Sorry for off-topic question. Does anyone have experience of the stabilisation of water-soluble proteins by detergents? Protein I'm working with is definitely water-soluble and has high yield, but, unfortunately, not very stable. Especially during concentration. So, we thought that adding some detergents may one of the ways to stabilise protein. So, did anyone do it before or may be know published examples? Any suggestions on the detergent type/concentration would be welcome. Thanks, Vitali
Re: [ccp4bb] CCP4 update 6.3.0 006
On Mon, Oct 08, 2012 at 06:20:59PM +, Ronan Keegan wrote: Dear CCP4 Users, A CCP4 update has just been released, consisting of the following changes: Hi Ronan et al, The update client on OS X doesn't seem to like our installation and dies with: Can't make class cfol of alias programs:i386-mac:ccp4:6.3.0:lib_exec:Update.app: into type Unicode text But I found an odd workaround. If I double-click the Update.app in Finder, I get the administrator password prompt, enter the credentials, and then the updater tells me that $CCP4 is unset, etc. I can then run 'open Update.app' from the shell, and it inherits $CCP4 and runs correctly. Any ideas? The workaround works, but since I don't really know why, I don't feel particularly good about it. Also, my installation is on NFS and is not owned by root, so it doesn't require administrator privileges to update. It would be nice if the application checked for write privileges before assuming it needs to be run with escalated privileges. -ben -- | Ben Eisenbraun | SBGrid Consortium | http://sbgrid.org | | Harvard Medical School | http://hms.harvard.edu |
